Despite advances in oligonucleotide therapeutics, the main issues remain cell delivery and cellular uptake. A number of strategies have been developed to combat this, the most widely used being the conjugation of a ‘delivery’ reagent to the oligonucleotide. In general the reagent is hydrophobic in nature, e.g. cholesterol, and is often attached via a cleavable linker. This is typically incorporated at the 5'-end of the oligo, and for siRNA is incorporated on the sense (passenger) strand. Cholesteryl-conjugated oligonucleotides have been the subject of substantial interest in antisense and other studies due to the lipophilicity and good availability of cholesterol. One such study ⁽¹⁾ has shown the use of cholesteryl-modified siRNA in therapeutic gene silencing. For 5'-attachment, we have found 5'-Cholesterol-CE Phosphoramidite (LK2170) to offer specific advantages in oligo synthesis. Since the cholesterol is attached directly to aminohexanol, it is not susceptible to 1,2-diol elimination as observed in some other products. Lack of a trityl group simplifies purification; some cholesterol products must be used in trityl-on mode (to prevent 1,2-diol elimination during deprotection), then detritylated, and can subsequently be very difficult to purify. Similarly, 5'-Cholesterol-TEG-CE Phosphoramidite (LK2189) is a simple 5'-modifier without the complications of a 1,2-diol and trityl protection, but with the added benefit of solubility in acetonitrile conferred by the TEG spacer. For 3'-modification we offer 3'-Cholesterol CPG 1000/110 (LK2394) and a 3'-Cholesterol TEG CPG, the latter in various pore sizes and functional loadings. LK2394 has an additional benefit in certain circumstances, since the modification is based on the natural sugar-phosphate backbone, hence there are no adverse structural effects on the oligo. The strict guidelines imposed by regulatory authorities now make it essential to use non-animal based products in pharmaceutical drug development for humans. With increasing frequency, therefore, our customers are requesting that we supply products with BSE/TSE statements. We have now developed an alternative route to these products that uses entirely plant-derived cholesterol, making them even better choices for modification of oligos.

Ref:

1. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs, J. Soutschek, A. Akinc, B. Bramlage, K. Charisse, R. Constien, M. Donoghue, S. Elbashir, A. Geick, P. Hadwiger, J. Harborth, M. John, V. Kesavan, G. Lavine, R.K. Pandey, T. Racie, K.G. Rajeev, I. Röhl, I. Toudjarska, G. Wang, S. Wuschko, D. Bumcrot, V. Koteliansky, S. Limmer, M. Manoharan and H.-P. Vornlocher, Nature, 432, 173-178, 2004.